The overall objective of this proposal is to identify novel antileukemic drugs for treatment of acute myeloid leukemias (AMLs) via activation of the NR4A subclass of orphan nuclear receptors. AMLs are primarily diseases of corruption of normal transcriptional control of myeloid cell development leading to accumulation of immature blasts and emergence of a transformed leukemic initiating cell (LIC) population with long term self renewal properties capable of sustaining leukemic expansion. NR4A1 and NR4A3 are potent tumor suppressors of AML. They are silenced in all human AML LICs irrespective of patient cytogenetics. Deletions in mice lead to extremely rapid postnatal AML due to disruption of hematopoietic stem cell (HSC) homeostasis and the emergence of a transformed radio-resistant LIC. Further, acute rescue of NR4A1 or NR4A3 in human AML cells inhibits their proliferation and reprograms a subset of gene signatures that distinguish all primary human LICs from normal HSCs regardless of cytogenetics. We hypothesize that NR4A silencing is an obligate step in AML development and that strategies directed toward their reactivation may be of general therapeutic benefit in treatment of AMLs. By intersection of our NR4A target based genomics data with in silico chemical genomics screening, we have employed a novel integrative strategy to successfully identify small molecules that reactivate NR4As in AML cells and that can be used to disclose mechanisms of NR4A silencing. Our specific aims in this proposal are: 1) to further validate NR4As as novel therapeutic targets for treatment of human AMLs by analysis of the cellular and molecular consequences of NR4A reactivation in human AML cells, 2) to test the anti-leukemic properties of small molecule activators of NR4As in human AML cells by analysis of their efficacy in elimination of LICs using AML cell lines, primary cells from AML patients and animal models of human AML and by interrogation of their AML selectivity by comparison of their effects on normal human HSCs, and 3) to disclose mechanisms of silencing and reactivation of NR4As in AML cells. In this aim, we will combine informatics pathway analysis of small molecule activators with molecular analysis of the chromatin landscape of the NR4A genes to identify marks and mediators of NR4A silencing in AML and to disclose signaling pathways involved in their reactivation. Given the widespread silencing of NR4As in AML patients and the sufficiency of their inactivation in causing AML, we believe that a successful outcome from these studies in terms of new drug discovery will quickly result in new clinical trials that will have major impact on treatment of AML patients.